PRÓPOLIS VERDE BRASILEIRA E TERAPIA FOTODINÂMICA ANTIMICROBIANA: CARACTERIZAÇÃO, AVALIAÇÃO IN VIVO E DESENVOLVIMENTO DE MICROEMULSÃO PARA O TRATAMENTO DE INFECÇÕES INTRADÉRMICAS CAUSADAS POR Staphylococcus aureus

Abstract

Introduction: Skin and soft tissue infections are an important global public health problem. Staphylococcus aureus is the main cause of this type of infection, so the development of resistance is one of the main factors linked to its spread and the challenges in its treatment. In this context, antimicrobial photodynamic therapy (TFA) with natural products has proven to be a good alternative in combating infections caused by antibiotic resistant microorganisms, as they add benefits inherent to these compounds to the damage caused by reactive oxygen species induced by TFA. Brazilian green propolis has known effects as an antimicrobial and immunomodulatory agent. However, it is not known for certain whether such activities would be maintained if used in the context of TFA. Objective: The objective of the present study was to characterize the phytochemical profile of Brazilian green propolis and its activity as a photosensitizer for TFA, evaluate its antimicrobial and immunomodulatory effects in vivo and develop microemulsion for the treatment of intradermal infections caused by antibiotic-resistant S. aureus. Materials and Methods: UV-Vis molecular absorption spectrometry was performed to evaluate the wavelengths with the potential to activate green propolis. Subsequently, reference strains of Methicillin-Resistant Staphylococcus aureus (MRSA ATCC 43300) and Vancomycin Intermediate Staphylococcus aureus (VISA ATCC 700699) were exposed to varying concentrations of green propolis: 1µg/mL, 5µg/mL, 10µg/mL, 50µg/mL mL and 100µg/mL and stimulated by blue, green or red LED light. Next, the zeta potential of bacteria exposed to Brazilian green propolis was evaluated in order to characterize a possible site of propolis action. Soon after, high-performance liquid chromatography was performed with a diode array detector coupled to tandem mass spectrometry, together with classical analysis of molecular networks, to identify potential bioactive molecules with photodynamic activity. Finally, cytotoxicity analyzes were performed on erythrocytes and nucleated cells. After this step, the antimicrobial and immunomodulatory effects of using Brazilian green propolis as a photosensitizer against S. aureus in vivo were evaluated, in an intradermal infection model in mice. Finally, a 11 microemulsion was developed as a transdermal delivery system for Brazilian green propolis, characterizing the microemulsified system and carrying out preliminary stability tests. Results: Brazilian green propolis was observed to exhibit a pronounced absorption peak and increased photoresponsiveness when exposed to blue light in the range of 400nm and 450nm. This characteristic reveals significant photodynamic activity against MRSA and VISA at concentrations from 5 µg/mL. Furthermore, propolis comprises compounds such as curcumin and other flavonoids from flavones, which have the potential for photodynamic activity and other antimicrobial functions. Tests with zeta potential did not show significant differences when the bacteria were exposed to Brazilian green propolis, which suggests potential internalization of the compound by the bacteria. Furthermore, Brazilian green propolis did not present toxicity to erythrocytes and nucleated cells. In vivo, TFA with Brazilian green propolis reduced the bacterial load at the site of infection, inhibited weight loss resulting from infection and modulated the inflammatory response through greater recruitment of neutrophil cells to the infected tissue. Furthermore, TFA induced an increase in the cytokines IL-17A and IL-12p70 in the draining retromaxillary lymph node. Finally, the microemulsion with Brazilian green propolis showed promising particle size, polydispersity index and zeta potential, as well as good stability in preliminary tests, maintaining the antimicrobial photodynamic activity induced by blue light. Conclusions: Brazilian green propolis presents photodynamic activity, when stimulated by blue LED light, capable of inducing the death of different strains of S. aureus, reducing bacterial load and modulating the inflammatory response in a murine model of intradermal infection. Furthermore, a microemulsion with Brazilian green propolis shows promise, as it presents interesting physical-chemical characteristics and stability, enabling the development of protocols that do not require local injection of the photosensitizer, without generating pain and discomfort, which can reduce therapeutic abandonment and increase adherence to treatment.